The induction of proline oxidase accompanying p53-dependent induction of apoptosis suggests that the proline metabolic pathway plays a role in programmed cell death. We are studying this metabolic pathway at the level of : a) proline oxidase - its induction and its role in apoptosis; b) pyrroline 5-carboxylate (P5C) and P5C reductase in cell signaling; and c) the function of prolidase in cell physiology. a) Proline oxidase - its induction and its role in apoptosis. In cells undergoing apoptosis, proline oxidase is induced and added proline stimulates the formation of reactive oxygen species (ROS). Importantly, in p53 negative cells transfected to overexpress proline oxidase, the addition of proline is sufficient to induce apoptosis. To define the specific reactive oxygen species, we co-expressed Ad-MnSOD, Ad-CuZnSOD or Ad-catalase in cells expressing POX. These experiments showed that POX generates superoxide radicals which is the mediator of apoptosis. Hydrogen peroxide, on the other hand, appears to be antiapoptotic. POX expression not only activates the intrinsic mitochondrial apoptosis pathway, but also the extrinsic, death receptor pathway. Recently, it has been shown that the expression of proline oxidase is silenced in certain tumors in the face of normal expression of p53, p21 and BAX suggesting that proline oxidase may act as a cancer suppressor. We are characterizing the proline oxidase promoter to determine its regulation on a molecular level. PPARgamma and its ligands are very active in upregulating POX expression. In addition, we are monitoring the expression of genes participating in this metabolic paradigm using real-time PCR and panels of cancer cells and tumors. There is differential expression of the two oxidase enzymes, PRODH which codes for proline oxidase and PRODH2 which codes for hydroxyproline oxidase. PRODH and PRODH2 are expressed at high levels in renal tissue. In contrast, several tumor cell lines have low expression of both oxidase genes. Finally, we are investigating the role for this metabolic pathway in the hypoxic response which is known to be regulated by the prolyl hydroxylation of HIF-1 alpha. P5C is an inhibitor of HIF-1 alpha prolyl hydroxylase and expression of POX activates the binding of HIF-1 alpha to its response element. In summary, these studies show that proline and pyrroline-5-carboxylate may be important in metabolic signaling in development, proliferation, and apoptosis. b) Pyrroline 5-carboxylate (P5C) as a signaling and regulatory molecule. P5C, the product of proline oxidase, ornithine aminotransferase and glutamate synthase, has been shown to have regulatory activities. Recent work suggests that P5C reductase (P5CR) may play a role in receptor-mediated regulation. The two known isozymes of P5CR may serve distinct functions. P5CR1 is a NADH-dependent enzyme regulated to produce proline, whereas P5CR2 is a NADPH-dependent enzyme coupled with redox-related regulation. In lymphocytes activated by lectins, it appears that P5CR2 is induced. We are defining the interaction of P5CR2 with membrane receptors. c) Inhibition of prolidase by nickel. Prolidase hydrolyzes imidodipeptides with carboxyl terminus proline and is a metalloenzyme. Nickel which can cause pulmonary fibrosis and cancer in humans, is a potent inhibitor of prolidase activity. Kinetic studies show that nickel is a competitive inhibitor of the interaction with manganese, the most potent activator of catalytic activity. The inhibitory effect can be demonstrated not only in cell-free systems, but also in intact CHO-K1 cells which are proline auxotrophs but which also can grow on GLY-PRO. Nickel markedly inhibits growth of these cells on GLY-PRO but not on PRO. We are using these cells to perturb the processing of imidodipeptides and other proline containing peptides to investigate their potential role in cell signaling.